Attenuated live rubella virus vaccine and method of production

ABSTRACT

Rubella virus is passaged in primary rabbit kidney tissue at least 15 times, preferably 51-61 times, at temperatures of 28*36* C, to obtain an effective vaccine. Test data are disclosed.

United States Patentv Huygelen et al.

1 51 Aug. 22, 1972 1541 ATTENUATED LIVE RUBELLA VIRUS VACCINE AND METHOD OF PRODUCTION [72] Inventors: Constant Huygelen, Vossekoten, Huldenberg; Julien Peetermans, 6, Avenue J. Herman, Rixensart, both of Belgium 122 Filed: 01.11 1967 [21] Appl.No.: 674,650

[51] Int. Cl. ..Cl2k 5/00, C12k 7/00 [58] Field of Search ..424/89;-195/l.3; 167/78 [56] 1 References Cited UNITED STATES PATENTS 3,401,084 9/1968 Buynak et a1 ..195/1.3

FOREIGN PATENTS OR APPLICATIONS 4,946,664 3/1966 Australia OTHER PUBLICATIONS Belcourt et al., Canadian Journal of Public Health, Vol. 55, pages 532- 534. December 1964. Public Health Reports, V01. 81, page 614, July 1966.

Primary ExaminerRichard L. Huff Attorney-William H. Edgerton, Richard D. Foggio,

Joan S. Keps, Arthur R. Eglington and Alan D. Lourie [57] ABSTRACT Rubella virus is passaged in primary rabbit kidney tissue at least 15 times, preferably 51-61 times, at temperatures of 28-36 C, to obtain an effective vaccine.

Test data are disclosed.

10 Claims, No Drawings A'I'IENUATED LIVE RUBELLA VIRUS VACCINE AND METHOD OF PRODUCTION This invention relates to an attenuated live rubella virus vaccine capable of inducing active immunity against rubella and to a process for producing such vaccine.

The term attenuated live virus employed in this specification refers to a rubella virus (RV) strain, the virulence of which has been attenuated by at least serial passages on primary rabbit kidney tissue cultures.

According to the procedure described hereafter in this specificatiomthere is obtained a modified virus and thereby a vaccine presenting high antigenicity while producing no appreciable undesirable reaction when inoculated to children.

The presence of undesirable reactions and, among them, more particularly the possible dissemination of the'virus by the vaccinated people is one of the major problems in the development of a rubella live type vaccine.

It is known that besides complications such as encephalitis and thrombocytopenic purpura which are I severe but quite rare and also arthritis which is not uncommon but generally self-limited and of short duration,- the most dramatic complication of this disease is of teratogenic nature. Rubella indeed is a serious problem for women in the first trimester of pregnancy because of its incidence on congenital malformations in the fetus stillbirths or abortions.

In this prospect, it is essential that a child who receives a rubella live-type vaccine does not constitute a potential danger to any pregnant woman with whom he might come into contact.

Up to now, the only possible prevention means against such fetal rubella complications were either administration of gamma globulin in large doses to exposed pregnant women or exposure of young girls to rubella before they reach the child-bearing age.

The administration of gamma globulin is far from being considered by each specialist as an efiective treatment and, for obvious reasons, the exposure to the disease atan earlier age is not an acceptable answer to the problem.

It has now surprisingly been found that serial passages of rubella virus in primary rabbit kidney (RK) tissue cultures not only involves attenuation of the virulence but also provides a method for preparing an attenuated live rubella virus vaccine presenting no appreciable undesirable reactions.

By the term no appreciable undesirable reactions as cited above, it is understood not only that the inoculation of the vaccine according to this invention stimulates immunity without inducing the usual pathological symptoms of regular rubella but also that in the clinical trials conducted with a vaccine according to this invention, no serological or virological evidence of infection has been detected in susceptible intimate contacts.

Thus, it is an object of the present invention to provide a method for attenuating the virulence of rubella virus without loss of antigenicity, said method consisting in passaging serially a rubella virus strain at least 15 times on primary rabbit kidney tissue cultures.

It is a further object of the present invention to provide (l) attenuated live rubella virus obtained by said attenuation method and (2) a vaccine active against rubella, comprising as active ingredient attenuated rubella virus obtained bysaid attenuation method.

. In other words, the present invention affords a method for preparing a vaccine active against rubella, said method consisting in passaging serially a rubella virus strain at least 15 times on primary rabbit kidney tissue cultures and using the harvested attenuated rubella virus as active ingredient for a rubella vaccine, according to any technique known to the art for vaccine formulation.

According to this invention, rubella virus undergoes a sufficient number of passages in primary rabbit kidney tissue cultures until attenuation is obtained. It has been found that attenuation requires at least 15 passages and preferably from 20 to 60 passages, said passages being conducted at a temperature not exceeding 36C.

According to a preferred embodiment of this invention, the duration of each passage is comprised between 3 and 6 days.

A vaccine is then prepared from an appropriate passage in primary RK cells using any technique known to the art for such preparation.

The rubella virus employed for carrying out this invention is isolated from a typical clinical case, using for instance throat swabs or urine or gargle samples. The samples are either frozen immediately and maintained at -60C until used or immediately inoculated into an adequate tissue culture system, e.g., primary African green monkey kidney (GMK) monolayers or any other tissue culture system known to the art for such isolation.

The presence of rubella virus is checked by challenging cultures for instance with an enterovirus such'as Echovirus l l or Coxsackievirus A 9 on the 9th or 10th day after inoculation.

Using specific antiserum prepared in rabbits against a known RV strain, it is possible to identify rubella virus by specific neutralization test in susceptible cells such as GMK cells. The absence of adventitious simian viruses is checked in- GMK cells after neutralization by specific RV antiserum prepared in an other cell system.

The primary rabbit kidney cell cultures for the serial passages are preferably prepared from kidneys of animals not older that 3 weeks old. A preferred growth medium for primary RK cultures is Hanks balanced salt medium supplemented withinactivated calf serum, lactalbumin hydrolysate and tryptose phosphate broth but other media known by those skilled in the art can also be employed. The incubation temperature is not above 36C. The serial passages are carried out by inoculating RK monolayers with aliquots of the supernatant fluid from the previous passage and preferably harvesting the virus between the 3rd and the 6th day after inoculation. Titration of the harvested virus can be carried out using the interference method in GMK culture tubes, using for instance Echovirus 11 or Coxsackievirus A 9 as indicated above for the isolation step.

At a certain passage level, not before the 15th and preferably between the 20th and the 60th passage, the supernatant fluid of the inoculated cultures is discarded by the 6th day after inoculation and the monolayers are washed with a bufiered saline solution, for instance Eagles solution or Hanks solution and further incubated with a maintenance medium, e.g., Hanks medium supplemented with casein hydrolysate. After a further incubation for 3 days, the supernatant fluid is harvested and clarified by filtration or by centrifugation.

Alternatively, harvesting of the virus is carried out after freezing, thawing and shaking of the culture in the maintenance medium and subsequent centrifugation or filtration.

The harvested RV supplemented or not with a stabilizing additive may be stored either in the frozen state, for instance at about 60C or in the freeze-dried state. The obtained vaccine is administered by subcutaneous or intramuscular route, if necessary after reconstitution.

The following examples illustrate the invention, they should not be construed as limiting the scope of the invention.

EXAMPLE 1 A 3-week-old rabbit from a breeding colony is examined for absence'of pathological lesions and sacrificed. Both kidneys are aseptically removed and cut into little pieces which are brought into contact with a buffered saline solution of trypsin (2.5 g/l.) and the mixture is continuously stirred for 10 minutes at a temperature of 37C. The liquid is then poured off and replaced by an equal volume of fresh trypsin solution. Trypsinization is then continued while. stirring until exhaustion of the tissue, the cells suspended in the liquid being removed from time to time and then centrifuged.

The cell sediment obtained is resuspended in Hanks solution and again centrifuged. This last step is repeated twice and the final cell sediment is suspended in growth medium consisting of Hanks balanced salt medium with 10 percent inactivated calf serum, 0.5 percent lactalbumin hydrolysate and 0.1 percent tryptose phosphate broth so as to secure a concentration of about 10 cells per ml.

Aliquots (1 ml) of the obtained cell suspension containing each about cells, are poured into 10 culture tubes (12 mm diameter) which are incubated in a slightly slant position at 37C for 4 days. After that incubation period, a complete monolayer is developed. The nutrient medium is then replaced by an equal volume of fresh one just before virusinoculation.

The same nutrient medium is used before and after virus inoculation and each primary RK cell culture indicated in this example is prepared according to the hereinabove described technique.

Aliquots (0.1 ml) of a rubella virus strain isolated in primary GMK cultures and passaged three times in this system for further characterization is inoculated into primary RK culture tubes which are incubated at 34C on a slant position.

On the sixth day after inoculation, the supernatant fluid is harvested and the harvested virus is identified and titrated using the interference method described above.

The titer in GMK cells after this first passage on RK cells is 10 InDm per ml.

Aliquots of the pooled supernatant fluid from the first passage are inoculated into other primary RK culture tubes which are incubated at 34C.

On the sixth day after inoculation, the supernatant fluid is harvested. The virus is titrated as indicated at the end of the first passage. This procedure is repeated up to the th passage, the incubation period of each individual passage ranging between from 3 to 5 days.

From the 9th passage onwards, a cytopathic effect is observed in primary RK cells.

For the 21st passage in RK cells, primary RK cell cultures are prepared in 500 square centimeter Roux flasks, using the technique described above.

After an incubation period of 3 days at 36C, a complete monolayer is obtained. The supernatant fluid is then discarded and replaced by an equal volume of the same growth medium and inoculated with aliquots of the viral material from the 20th passage.

After further incubation for 6 days at 34C, the supernatant fluid is discarded and replaced by an equal volume of maintenance medium consisting of Hanks solution supplemented with 0.3 percent casein hydrolysate. After further incubation for 3 days at 34C, the supernatant .fluid' is harvested and clarified by centrifugation.

Samples are taken for titration, identification and safety testing and the virus is stored at 60C.

The virus titer is 10 InD per ml as tested in primary GMK cells by the interference system.

This virus material is then subjected to safety testing including bacterial sterility and absence of adventitious agents by inoculation into rabbits, hamsters, guinea pigs, mice and monkeys.

Additional safety testing is performed in different tissue culture systems.

Preliminary potency testing is performed by inoculating rabbits and monkeys with 10 InD The serumneutralization (SN) tests are performed in GMK cells.

Results are given in the following Tables I and 11.

TABLE I Antibody response of monkeys inoculated intramuscularly with 10 InD Preinoc- Monkey ulation Results of SN test against 30 lnD n serum sample in GMK cells 14' 25' 32* 45" 225 16 ND 16 1/16 1/16 509 14 'ND V4 1/16 1/32 611 16 14 1G ND 1/32 831 /4 1/32 ND 1/32 619 14 )4 A ND 1/16 857 14 14 A ND 1/16 1064 14 ND 1/32 ND 1/64 953 14 ND 1/16 ND 1/32 1053 /4 ND 14 1 ND 14;

ND Not determined days after inoculation TABLE II Antibody response of rabbits inoculated subcutaneously with l0 lnD Preinoc- Rabbit ulation Results of SN test against 30 lnD in GMK cells 24 days after inoculation n serum sample 1 The virus preparation is divided into glass vials each of them containing 1 ml of fluid.

The vials are then freeze dried and sealed.

After reconstitution by adding 1 ml of distilled water, the vaccine is inoculated by subcutaneous route to susceptible subjects, the individual doses being about 125 InD Results of a serological response testing conducted in a group of 25 seronegative children (15 receiving vaccine and controls living in close contact) are given in Table III.

From the vaccinated children, 13 had a rise of antibodies up to 1/32 or more (two samples-indicated ND-were not available at the time of testing). None showed clinical signs of infection. All 10 contacts remained serologically negative.

TABLE III Serological response expressed in seroneutralizing antibodies titer.

prevac- Subjects age (years) cination status 56 days after sample vaccination S.M. 2% 4 V 32 vs. 2 4 V 32 MJ. 2 4 V 32 N.R. 2 4 V '32 H.A. 1 4 V 32 DB.G 2 4 C 4 VN.L 2 4 C 4 RF. 2 4 V 32 BD. 2 4 C 4 .|.A. l% 4 C 4 V.M. 1 4 V 32 BF. 1 4 V 32 LI. 1 4 C 4 CC. I 4 V 32 A.M. 1 4 V 32 RD. I 4 C 4 LA. 2 4 V ND D.A. 2 4 C 4 6.0. 2% 4 C 4 M.A. 2% 4 V ND LA. 1 4 C 4 RA. 2% 4 V 32 DB. 3% 4 V 32 GB.]( 5 4 V 8 GB.F 4 4 C 4 V= vaccinated C= contact EXAMPLE 2 The technique is that described in Example 1 but the passages are continued up to the30th passage in primary RK cell cultures.

A vaccine is prepared therefrom and safety testing is performed as described in Example 1.

The potency of the preparation is tested in animals (monkeys).

Results are given in Table IV.

TABLE IV Antibody response of monkeys inoculated intramuscularly with 10 lnD Monkey Preinoculation Results of SN test against 10 lnD in GMK cells n serum sample 39' 1098 V4 54; ND

943 ll l6 l/ l 6 days after inoculation EXAMPLE 3 Starting from the viral material from the 2 1 st passage at 34C obtained in Example 1, nine further serial passages are carried out in primary RK cell cultures but at 28C, including the last step for vaccine preparation.

The obtained vaccine is safety tested and inoculated into monkeys and rabbits for potency testing.

Results are given in the following Tables V and VI.

TABLE V Antibody response of monkeys inoculated intramuscularly with 10 'lnD Preinoculation Monkey Results of SN test against l0 lnD in GMK cells n serum sample 25* 39* 1003 V; l/ 16 977 /4 rt days after inoculation TABLE VI Antibody response of rabbits inoculated intramuscu- I larly with l0 lnD The technique is that described in Example 1 but the passages are continued up to the 51st passage in primary RK cell cultures, the final maintenance medium consisting of Hanks solution supplemented with 0.3 percent casein hydrolysate and containing 50 mcg of chloramphenicol per ml of Hanks solution.

After incubation for 9 days at 34C, the supernatant fluid is harvested, clarified by centrifugation and mixed with an equal volume of stabilizing solution consisting of 30 g of potassium glutamate, 200 g of sucrose and 50 mg of chloramphenicol per liter of distilled water and the mixture is distributed into glass vials containing 1 ml of water.

The vials are then freeze-dried and sealed.

After reconstitution by adding 1 ml of distilled water, the vaccine is inoculated by subcutaneous route to 65 seronegative subjects, the titration of the individual 'doses reaching about l0"-InD in GMK cells or 10" PFU (Plaque forming units) in RK 13 cells. A group of 30 seronegative children was kept in intimate contact with the vaccinees for a period of 6 weeks.

A serological response testing conducted in the group of 65 vaccinated subjects showed that all but one responded to the vaccine with a means antibody titer of 1/ 128 as assayed in HA! (Hemagglutination Inhibition) test. None showed clinical signs of infection. All 30 contacts remained serologically negative.

1 EXAMPLE 5 The technique is that described in Example 1 but the passages are continued up to the 61st passage in primary RK cell cultures, the final maintenance medium consisting of Hanks solution supplemented with 0.3 percent casein hydrolysat e and containing 50 mcg of chloramphenicol per ml of Hanks solution.

After incubation for 9 days at 34C, the supernatant fluid is harvested, clarified by centrifugation and mixed with an equal volume of stabilizing solution consisting of 30 g of potassium glutamate, 200 g of sucrose and 50 mg of chlora'mphenicol per liter of distilled water and the mixture is distributed into glass vials containing 1 ml of water.

The vials are then freeze-dried and sealed.

After reconstitution by adding 1 ml of distilled water, the vaccine is inoculated by subcutaneous route to seronegative subjects, the titration of the individual doses reaching about l0 -"InD in GMK cells or 10" PFU in RK 13 cells.

A serological response testing conducted in a group of seven seronegative subjects showed that all responded to the vaccine with a mean titer of 1/64 as assayed in HAI test None showed clinical signs of infection.

What we claim is:

1. A method for attenuating the virulence of rubella virus without loss of antigenicity comprising passaging serially a rubella virus strain for 3 to 6 days 51 to 61 times in primary rabbit kidney tissue cultures at 28-36 C 2. A method accordingto claim 1 wherein the passages are each conducted at 34 for 3. to 5 days.

3. A method according to claim 2 wherein the number of passages is 51.

4. A process for attenuating a live rubella virus comprising serially passaging a rubella virus strain, after isolation and characterization, solely in primary rabbit kidney tissue culture from 51-61 times inclusive for 3 to 6 days at about 34 to produce an antigenically active, non-communicable, minimal side effect-producing, live rubella virus.

5. A process for producing an attenuated live rubella virus which comprises the steps of introducing a virulent, live rubella virus into a rabbit kidney tissue cell culture, incubating said tissue culture at a temperature compatible with growth of said tissue and said virus, harvesting at least a portion of the virus so produced, reintroducing said harvested virus into fresh culture of the tissue, and repeating such tissue culture passage for at least 50 passages to produce an antigenically active, non-communicable live rubella virus.

6. A method for preparing a rubella vaccine comprising passaging serially a rubella virus strain for 3 to 6 days 51 to 61 times in primary rabbit kidney tissue cultures at 2836C, and then combining the resulting attenuated virus with a vehicle.

7. A rubella vaccine prepared according to the process of claim 6.

8. A method according to claim 6 in which the passages are each conducted at 34 for 3 to 5 days.

9. A method according to claim 8 wherein the number of passages is 5 l.

10. A method of preparing an attenuated rubella tissy ssss an; t astz zias"ssssws s been attenuated by passage of at least 50 times in rabbit kidney tissue culture, said further incubation being for a period of time suflicient to pennit growth of a larger amount of said virus, and harvesting the resulting virus material.

UNITED STATES PATENT OFFICE PU-IOSO 5 69 CERTIFICATE 9F CORECTION Dated August 22, 1972 Patent No. 3,686,394

Inventofls) Con a t I It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 1, after line reading "of Belgium" insert Assignee: Recherche et Industrie Therapeutiques, R.I.T.,

Genval, Belgium Signed and sealed this 9th day of January 1973.

(SEAL) Attest: EDWARD M.FLETCHER JR. ROBERT GOTTSCHALK Attesting Officer Commissioner of Patents 

2. A method according to claim 1 wherein the passages are each conducted at 34* for 3 to 5 days.
 3. A method according to claim 2 wherein the number of passages is
 51. 4. A process for attenuating a live rubella virus comprising serially passaging a rubella virus strain, after isolation and characterization, solely in primary rabbit kidney tissue culture from 51-61 times inclusive for 3 to 6 days at about 34* to produce an antigenically active, non-communicable, minimal side effect-producing, live rubella virus.
 5. A process for producing an attenuated live rubella virus which comprises the steps of introducing a virulent, live rubella virus into a rabbit kidney tissue cell culture, incubating said tissue culture at a temperature compatible with growth of said tissue and said virus, harvesting at least a portion of the virus so produced, reintroducing said harvested virus into fresh culture of the tissue, and repeating such tissue culture passage for at least 50 passages to produce an antigenically active, non-communicable live rubella virus.
 6. A method for preparing a rubella vaccine comprising passaging serially a rubella virus strain for 3 to 6 days 51 to 61 times in primary rabbit kidney tissue cultures at 28*-36*C, and then combining the resulting attenuated virus with a vehicle.
 7. A rubella vaccine prepared according to the process of claim
 6. 8. A method according to claim 6 in which the passages are each conducted at 34* for 3 to 5 days.
 9. A method according to claim 8 wherein the number of passages is
 51. 10. A method of preparing an attenuated rubella virus vaccine comprising further incubating in rabbit kidney tissue culture a rubella virus that already has been attenuated by passage of at least 50 times in rabbit kidney tissue culture, said further incubation being for a period of time sufficient to permit growth of a larger amount of said virus, and harvesting the resulting virus material. 